Activating Notch mutations are found in 60% of human T cell acute lymphoblastic leukemia cases (T-ALL). A direct transcriptional target of Notch is the cell cycle regulator Myc, which is the most frequently amplified oncogene across a wide range of malignancies. In the proposed work, I will characterize Myc transcriptional regulation in T-ALL. We have previously identified an enhancer region termed the Notch-Dependent Myc Enhancer (NDME) that is active in a reporter assay and loops to the Myc promoter in mouse and human T- ALL cells. Four T cell transcription factors bind in this region, but their role in activating the NDME is unknown. Previous work from our lab showed that T-ALL cells created to be resistant to Notch inhibition maintain Myc expression through a switch in enhancer usage to another region, termed the Brd4-Dependent Myc Enhancer (BDME). A potential treatment for T-ALL is the BET family inhibitor JQ1, which has already proved efficacious in AML. I have generated JQ1-resistant human T-ALL cells that maintain MYC expression. In Aim 1, I will determine the roles of four T cell transcription factors, Notch1, E2A, Gata3, and Runx1 in activating the NDME in T-ALL. I will specifically target the Notch1 binding site in the NDME using CRISPR/Cas9 technology in T-ALL cell lines. I will assay the activity of the NDME using growth curve analysis and local ChIP for histone marks of activation. In addition, I will perform chromatin looping studies to determine the effects of loss of factor binding on looping of the NDME to the Myc promoter. This will determine the importance of T cell-specific factors in regulating Myc expression through the NDME. In Aim 2, I will determine the mechanism of MYC expression in JQ1-resistant human T-ALL cells. In nave T- ALL cells, both the NDME and the BDME loop to the Myc promoter. I hypothesize that in contrast to the GSI- resistant cells previously characterized, these cells will use only the NDME and not the BDME to drive Myc expression. BET family members are also crucial for the activation of a class of enhancers termed super or stretch enhancers. These super enhancers often regulate key oncogenic targets. I will also determine the activation status of T cell-specific supe enhancers in JQ1-resistant T-all cells. This work will provide important knowledge into mechanisms of JQ1 resistance in T-ALL. This work will provide critical insights into the mechanisms of regulation of the gene Myc and may identify novel targets for treatment of both neoplastic de novo and refractory T-ALL.